Olivier Hyrien

Eukaryotic Chromosome Replication

Our long-term goal is a quantitative understanding of whole-genome replication in multiple eukaryotes.

Research context and recent results

DNA replication is tightly regulated to accurately duplicate the genome despite endogenous and exogenous obstacles. Replication origins are "licensed" in G1 for use in the next S-phase by loading the replicative helicase in an inactive form around DNA. During S phase, protein kinases and accessory factors cooperate to activate the helicase and assemble bidirectional replisomes. Only a fraction of the helicase complexes are activated during S phase. Redundant complexes provide backup, potential origins that can rescue replication downstream of stalled forks and boost S phase completion.

Despite decades of investigation, the nature of mammalian origins is still unclear (3). Using genome-wide replication timing profiles and single-molecule replication mapping by DNA combing, we identified in the human genome about 1,500 megabase-scale domains with a U-shaped replication timing profile and a predicted N-shaped replication fork directionality (RFD) profile (4-7). Recently, we determined RFD genome-wide by massive sequencing of highly purified Okazaki fragments (3 ; Fig. 1&2). The results strikingly confirmed the predicted N-shaped RFD gradients and allowed us to map replication initiation and termination zones with unprecedented clarity and resolution (3). We propose that replication first initiates at efficient master initiation zones, detected by abrupt RFD inversions, and then propagates between them by activation of more dispersed, less efficient origins. Numerical and analytical models showed an excellent agreement between theory and these data (2,4). Definitive proof will require visualisation and quantification of secondary origin activation by high-througput single-molecule analyses, a topic of ongoing technological development (1).

Projects

Most DNA replication mapping techniques only provide an average picture of genome replication in a cell population, whereas different chromosomal copies replicate differently due to stochasticity in replication initiation and fork progression. Single-molecule methods are required to evaluate cell-to-cell heterogeneity, to reveal rare but important events (e.g. pathological fork stalling) hidden in population averages, to study correlations between neighboring replication units. Such information is key to correct mathematical modelling of whole-genome replication (3,5). Unfortunately, current single-molecule methods are of very low throughput. We are developing novel techniques to simultaneously visualize in a single day the entire length, replication tracts, and restriction map of tens of thousands of stretched DNA molecules (Fig. 2). This huge increase in throughput and image quality, combined with automated image analysis and mapping, will allow to quantify replication initiation, elongation and termination genome-wide at the single-molecule level in yeast, Xenopus and normal or cancer human cells. The results will instruct advanced mathematical modelling and reveal how replication parameters relate to chromatin structure, transcription and genomic instability in the three organisms, which will lead to a more unified view of eukaryotic DNA replication.